Lv PEPTIDE, ANTI-Lv ANTIBODY AND METHODS THEREOF

ABSTRACT

Embodiments of the invention are directed to the administration of the composition containing a portion of the peptide Lv to a subject to promote angiogenesis. The interaction between peptide Lv and VEGFR2 represents a novel pathway regulating angiogenesis and cardiac function. The artificially modified peptide Lv, the inverso D-peptide Lv, shows a similar efficacy in promoting endothelial cell proliferation as the natural peptide Lv. Additional embodiments are directed to the use of anti-Lv antibodies for reducing angiogenesis and dampening L-VGCC activities.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to and incorporates byreference the entire disclosure of U.S. Provisional Patent ApplicationNo. 62/291,283 filed on Feb. 4, 2016.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made by Government support under contractsR01EY017452 and R21EY023339 awarded by The National Eye Institute of theNational Institutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Peptide Lv is a bioactive peptide that was identified throughbioinformatics screening. Peptide Lv is a short peptide with only 40amino acids in humans (49 amino acids in mice) and a ˜5.8 kD molecularweight. It can be applied to various biomedical processes that involvenew blood vessel formation (angiogenesis), such as, for example, woundhealing and cardiovascular repairing after ischemia injuries and/ordisease insults. It can also be used for forming new lymphatic vessels(lymphangiogenesis) and enhancing immunity, metabolism, and homeostasis.The molecular mechanism of peptide Lv on angiogenesis andlymphangiogenesis is that peptide Lv is able to activate vascularendothelial growth factor (VEGF) receptors and promote proliferation ofendothelial cells that are required for angiogenesis, lymphangiogenesis,and cardiovascular repairing. Peptide Lv is like VEGF and is able todilate blood vessels as a vasodilator, and thus it can be used toenhance cardiovascular function, decrease blood pressure, or treatmicrovascular insufficiency associated with coronary artery diseases. Inaddition, peptide Lv is able to enhance physiological functions ofneurons and cardiomyocytes by augmenting the L-type voltage-gatedcalcium channel (L-VGCC) currents, since L-VGCCs are essential in thecontraction of cardiomyocytes and neurotransmitter release from neurons.Therefore, peptide Lv can be used to enhance cardiac contraction,cardiac output, and neural function.

The antibody specific against peptide Lv (anti-peptide Lv antibody) canspecifically block the effect of peptide Lv, VEGF, or VEGF receptoractivation. Anti-peptide Lv antibody can be used to stop diseases thathave pathological angiogenesis and lymphangiogenesis or L-VGCChyperfunction, such as cancers, diabetic retinopathy, age-relatedmacular degeneration, cardiac arrhythmia, various neuropathy, andothers.

The main peptide Lv coding region contains 49 amino acids in mice and 40in human, which is conserved across human, mouse, rat, and chicken. Thepeptide Lv mRNA is widely expressed in various organs including thelung, spleen, intestine, retina, and various regions in the brain.However, the binding partners and receptors of peptide Lv are unknown,which completely cloud the physiological and pathological functions ofpeptide Lv. Since the brain expresses peptide Lv, a study applied aproteomics approach using a mouse whole brain preparation, andidentified that vascular endothelial growth factor receptor 2 (VEGFR2)is a binding partner of peptide Lv. VEGFR2 (KDR/FLK-1) belongs to atyrosine kinase receptor family that is subjected to autophosphorylationof tyrosine residues after the receptor is activated via ligand binding.VEGFR signaling is essential for the development of the cardiovascularsystem during embryonic stages, and it is critical for angiogenesis,lymphangiogenesis, vasodilation, and wound healing throughout adulthood.There are four major tyrosine autophosphorylation sites (951, 1054/1059,1175, and 1212/1214) located in the intracellular domain of VEGFR2.These four phosphorylation sites may elicit different signaling pathwaysupon activation, thus prompting different functions. Since VEGFR2 isimportant in the developing heart, the interaction between peptide Lvand VEGFR2 in the embryonic chicken heart was further verified. Thephysiological effects of peptide Lv in embryonic chicken cardiomyocytesand human endothelial cells was verified, and a potential functionallink between peptide Lv and VEGFR2 was found.

It has been shown that exogenous peptide Lv increases L-VGCC currents incultured retinal photoreceptors and cardiomyocytes. The augmentation ofL-VGCC currents is mainly through the increase of mRNA and proteinexpressions of L-VGCCα1 subunits. The L-VGCCs are highly expressed inthe nervous and cardiovascular systems. The voltage dependent calciumentry through L-VGCCs promotes various cellular processes includingregulating intracellular calcium homeostasis, modulating activities ofcalcium-dependent enzymes, and regulating gene expressions. Inparticular, L-VGCCs are essential for neurotransmitter release fromnon-spiking neurons, such as photoreceptors and bipolar cells in theretina. In brain neurons, L-VGCCs mediate activity-dependent calciuminflux, so L-VGCCs are critical in gating neuronal activities. WhileL-VGCCs are required for cognitive functions, learning and memory,dysregulation or hyperactivity of L-VGCCs causes epilepsy andaging-related memory loss. In cardiomyocytes, the L-VGCCs contribute tothe action potential and excitation-contraction coupling, anddysregulation of L-VGCCs in the cardiovascular system causes cardiacarrhythmias and heart failure. Since L-VGCCs are important for thefunction of cardiomyocytes, it was explored that peptide Lv regulatedcardiomyocyte physiology by augmenting the expression of L-VGCCs in partthrough VEGFR2-dependent signaling.

Peptide Lv can be used to promote: neurological function, vascularfunction, cardiac contractility and output, tissue repair and woundhealing, through regulating L-VGCCs, angiogenesis andlymphoangiogenesis, and neovascularization, while anti-peptide Lvantibody antagonizes those functions. Peptide Lv can also be used toreduce hypertension and improve blood flow to the ischemic tissues thatsuffer from microvascular spasm or occlusion through vasodilation. Byaugmenting L-VGCCs, peptide Lv can enhance/promote neural and cardiacelectrical transmission for treatment of neural/cardiovascular diseasesand heart failure related to the conduction deficiency. Anti-peptide Lvantibody can be used in treating cancers, diabetic retinopathy,age-related macular degeneration, and other diseases that are related topathological angiogenesis/lymphangiogenesis or L-VGCC hyperfunctions,such as cardiac arrhythmia, and various neural dysfunction andneuropathy (e.g. epileptic disorders, age-dependent memory deficits).

There are many diseases and conditions that are involved angiogenesis,lymphangiogenesis, or L-VGCC disorders. For example, wound healing andcardiovascular repairing require angiogenesis and lymphangiogenesis, sopeptide Lv can enhance these necessary biological processes. Thedeficiency in cardiac L-VGCC can lead to heart failure and certain formsof arrhythmia, so peptide Lv will be able to treat/prevent thesediseases. Pathological angiogenesis, lymphangiogenesis and L-VGCChyperfunction take place in many diseases such as cancer, diabeticretinopathy, age-related macular degeneration, cardiac tachycardia andvarious neural dysfunction and neuropathy (epileptic disorders,age-dependent memory deficits) and many more. Thus, anti-peptide Lvantibody can be used to stop or dampen pathological angiogenesis,lymphangiogenesis, and neural dysfunction. Therefore, both peptide Lvand anti-peptide Lv antibody will have great potential for treatingvarious medical conditions.

Applications related to VEGF and VEGF receptors are generally known.However, not all patients respond to VEGF or anti-VEGF treatments. Forexample, about 30% of patients with various retinopathies do not respondwell to anti-VEGF treatments to stop pathological angiogenesis. Somepatients further develop resistance to the current anti-VEGF treatmentsmonths after the treatments started. Peptide Lv is a molecule muchsmaller than VEGF. Its amino acid sequence is different from VEGF, so ifpatients do not respond to VEGF or anti-VEGF treatments, patients mayrespond to peptide Lv or anti-peptide Lv (anti-peptide Lv antibody)treatments. Peptide Lv and VEGF have synergistic effects, so peptide Lvor anti-peptide Lv may further enhance the efficacy of current existingVEGF or anti-VEGF therapies.

In addition to its angiogenic properties, peptide Lv also promotesL-VGCC currents, which are important regulators in cardiac and neuronalfunction. In some forms of heart failure and cardiac ischemia (lack ofblood flow), they are associated with reduced cardiac contractility andperfusion. Peptide Lv can promote cardiac angiogenesis and vasodilation(for providing more blood flow) and enhance cardiac L-VGCC currents(thus increasing cardiac contractility). These unique dual effects willbenefit the ischemic heart failure patients. Nearly 12% (1 million) ofall US patients with coronary artery diseases have diffuse vascularproblems (small vessel diseases), and the majority of them suffer frommicrovascular dysfunction, so the traditional treatments will not besufficient to treat such conditions. If left untreated, small vesseldiseases can lead to heart failure, myocardial infarction, and cardiacarrest. Thus, peptide Lv, with its angiogenic and vasodilationproperties, will be the important new agent in treating these coronaryartery diseases.

SUMMARY OF THE INVENTION

An embodiment of the invention is directed to the administration of thecomposition containing a portion of the peptide Lv to a subject topromote angiogenesis. Evidence of peptide Lv as an angiogenic agent isshown by its ability to promote endothelial cell proliferation andintra-ocular neovascularization.

In a further embodiment of the invention, the interaction betweenpeptide Lv and VEGFR2 represents a novel pathway regulating angiogenesisand cardiac function.

Another embodiment of the invention is directed to the use of anti-Lvantibodies for reducing angiogenesis and dampening L-VGCC activities.

An additional embodiment of the invention is directed to the compositionand use of an artificially modified peptide Lv, the inverso D-peptideLv, in which the first amino acid at the N-terminal is inverted from thenatural L-isomer to D-isomer. The D-peptide Lv shows a similar efficacyin promoting endothelial cell proliferation as the natural peptide Lv.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E, 1F and 1G show that peptide Lv enhances L-VGCCcurrents and protein expression in cultured embryonic cardiomyocytes andanti-peptide Lv antibody (a-peptide Lv) has an antagonist effect;

FIGS. 2A and 2B show that peptide Lv mediated enhancement of L-VGCCcurrents is blocked by VEGFR2 antagonist;

FIGS. 3A, 3B, 3C, 3D and 3E show that treatment of cells with peptide Lvelicits a significant increase of phosphorylated ERK levels andtreatment with a kinase inhibitor in the presence or absence of peptideLv decreased the current densities of L-VGCCs;

FIGS. 4A, 4B, 4C and 4D show that the endogenous agonist VEGFa mimicsthe effect of peptide Lv and enhances L-VGCC activity in chickenembryonic cardiomyocytes;

FIG. 5 shows that peptide Lv stimulates the cell proliferation of humanumbilical vein endothelial cells (HUVECs) in a dose-dependent manner;

FIGS. 6A, 6B and 6C show that peptide Lv elicits vasodilation, promotesendothelial migration for wound healing, and enhances VEGF's effect onendothelial proliferation;

FIGS. 7A and 7B show that peptide Lv promotes vascular growth and FIG. 9anti-peptide Lv antibody (anti-peptide Lv) dampens the vascular growth;

FIG. 8 shows that anti-peptide Lv antibody prevents pathologicalangiogenesis and neovascularization; and

FIG. 9 shows that anti-peptide Lv antibody dampens VEGF-elicitedendothelial cell proliferation in a dose-dependent manner.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

An embodiment of the invention is directed to a composition comprising aportion of peptide Lv. A further embodiment of the invention is directedto the administration of the composition containing a portion of thepeptide Lv to a subject to promote angiogenesis. Evidence of peptide Lvas an angiogenic agent is shown by its ability to promote endothelialcell proliferation and intra-ocular neovascularization. Directinjections of peptide Lv into vitreous significantly promote newgeneration of blood vessels (neovascularization). Further, peptide Lvcan promote wound healing by enhancing the proliferation and migrationof endothelial cells. In freshly isolated porcine retinal arterioles,peptide Lv increases the diameter of the blood vessels thus showing theability of peptide Lv as a vasodilator and potentially to decrease bloodpressure, as well as to promote microvascular circulation.

Peptide Lv is a peptide that enhances L-VGCC activities in conephotoreceptors through increased expressions of both mRNA and proteinlevels. Because L-VGCCs are essential for neurotransmitter release andactivity-dependent calcium influx in neurons, L-VGCCs are critical ingating neuronal activities and function. In the brain, L-VGCCs arerequired for cognitive functions, such as learning and memory. In theretina, L-VGCCs are essential for communications between neurons andtransmitting the visual signals from retinal photoreceptors all the wayto retinal ganglion cells and to the brain. The expression of peptide Lvcan be detected in various brain regions, including hippocampus,cerebral cortex, and cerebellum, which are all important for cognitivefunction and various learning and memories. All the major neurons in theretina, especially retinal photoreceptors express peptide Lv. Thus,peptide Lv may play a role in enhancing the function of L-VGCCs in theretina and brain.

VEGFR2 (KDR/FLK-1) is a potential receptor for peptide Lv, which is anunderlying mechanism for the augmentation of L-VGCCs by peptide Lv inthe cardiomyocytes. Peptide Lv is able to activate the phosphorylationof VEGFR2 (KDR/FLK-1) and its downstream signaling, while inhibition ofVEGFR2 (KDR/FLK-1) signaling blocked the actions of peptide Lv. Testresults suggest that peptide Lv can serve as a novel activator of VEGFR2through its augmentation of L-VGCCs in cardiomyocytes and its action onpromoting proliferation of endothelial cells. Furthermore, peptide Lvhas angiogenic properties and might play a regulatory role in thecardiovascular system.

In the cardiovascular system, peptide hormones, such as somatostatin,angiotensin II, and natriuretic peptides (BNP and CNP), are known to beinvolved in the regulation of heart rate, cardiac contraction, anddevelopment. Several VEGF family members, including VEGFa, VEGFb, VEGFc,VEGFd, platelet-derived growth factor (PDGF), and placental growthfactor (PLGF), can activate VEGFRs to regulate angiogenesis andlymphangiogenesis. Comparisons of the amino acid sequences of peptide Lvand the VEGF family have not revealed much homology. Through I-TASSER, apredicted secondary structure for peptide Lv was obtained and somesimilarities with VEGF members were found. Therefore, peptide Lv as anactivator for VEGFR2 may differ from other VEGF family members wheninteracting with VEGFR2. While at low concentrations which neither VEGFnor peptide Lv would have any effect, adding both VEGF and peptide Lvtogether cause a significant enhancement of endothelial proliferation.Thus, peptide Lv has a synergistic action with VEGF (FIG. 6). However,the mechanism by which peptide Lv interacts with VEGFR2 and promotesVEGFR2 dimerization remains a question.

Activation of VEGFR2 signaling elicits a rise of intracellular calciumconcentration through an increase of the intracellular calciumstore-related proteins or the conductance of transient receptorpotential (TRP) channels in various cell types. In cardiomyocytes,calcium influx through L-VGCCs triggers calcium release fromintracellular calcium stores, activates calcium-dependent kinases, andleads to excitation-contraction coupling. The VEGF-PLCγ pathway is knownto control cardiac contractility in the embryonic heart. Even thoughpeptide Lv has been shown to increase L-VGCC currents and the expressionof L-VGCCα1 subunits through the VEGFR2 signaling pathway incardiomyocytes, whether L-VGCCs are involved in the VEGF-mediatedcalcium-induced intracellular calcium release needs futureinvestigation. It is postulated that the augmentation of L-VGCC proteinexpression and currents by peptide Lv might lead to increasedcardiomyocyte contractions, because treatments with other neuropeptides,such as endothelin-1 and angiotensin-II, have been shown to enhancecalcium-dependent contraction in cardiomyocytes.

In summary, peptide Lv can interact with VEGFR2 and trigger receptortyrosine kinase activity, as well as its downstream signaling pathway incardiomyocytes. Peptide Lv enhances calcium entry by increasing L-VGCCcurrents through increasing the expression of L-VGCCα1 subunits. Theinteraction between peptide Lv and VEGFR2 represents a novel pathwayregulating angiogenesis and cardiac function.

Another embodiment of the invention is directed to an antibody that isspecific for a portion of the peptide Lv. The anti-peptide Lv antibody(anti-peptide Lv) decreases L-VGCC currents and blocks peptide Lv'sactions. While L-VGCCs are essential in various cardiac, brain andretinal functions, dysregulation or hyperactivities of L-VGCCs can causecardiac arrhythmia and epilepsy, and it may contribute to aging-relateddementia. Thus, these antibodies are capable of reducing angiogenesisand dampening L-VGCC activities, and can be used for the treatment ofdiseases that involve pathological angiogenesis and lymphangiogenesis,L-VGCC hyperfunction, or neural dysfunction. These diseases includecancers, diabetic retinopathy, age-related macular degeneration, cardiacarrhythmia, various neuropathies, and the like.

An additional embodiment of the invention is directed to the use of anartificially modified peptide Lv, the inverso D-peptide Lv, in which thefirst amino acid at the N-terminal is inverted from the natural L-isomerto D-isomer. The D-peptide Lv shows a similar efficacy in promotingendothelial cell proliferation as the natural peptide Lv.

WORKING EXAMPLES Chicken Embryonic Cardiomyocyte Culture

Fertilized eggs (Gallus gallus, Single Comb White Leghorns) wereobtained from the Poultry Science Department, Texas A&M University(College Station, Tex., USA). All chicken embryos were maintained at 39°C.±0.5° C. Chicken hearts were harvested at embryonic day 12 (E12) andventricular cardiomyocytes were dissociated and cultured onpoly-D-lysine/collagen double-coated dishes (for biochemical andmolecular biological assays) or coverslips (for electrophysiologicalrecordings) as described previously. All cultures were maintained at 39°C.±0.5° C. with 5% CO₂. Cells were cultured for 2-3 days and subjectedto treatments with vehicle, peptide Lv, or pharmaceutical blockers priorto Western blotting or electrophysiological recordings.

Reverse Transcription PCR (RT-PCR)

Total RNA from mouse eyes, spleen, intestine, lung, and heart wereisolated. One step RT-PCR amplification was used for detection ofpeptide Lv precursor and GAPDH mRNA expression as described previously.

Western Blots and Trichloroacetic Acid (TCA) Precipitation

Treated or control cells were washed and lysed inradioimmunoprecipitation assay (RIPA) buffer, and proteins weredenatured by mixing with 2× Laemmli sample buffer for 5 minutes at 95°C. Samples were separated on 10% sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulosemembranes. The primary antibodies used in this study were pan L-VGCCα1,ERK1/2, di-phosphorylated ERK1/2, actin, phosphorylated PKC α/βIIsubunit, phosphorylated tyrosine, VEGFR2, and phosphorylated VEGFR2 attyr1054/1059. Because chickens appear to express a single form of ERK(on the basis of molecular weight), the antibodies against ERK1/2 andpERK1/2 only label a single band on Western blots, while both antibodieslabel two bands in samples prepared from mammalian tissues. Blots werevisualized by the appropriate secondary antibodies and an ECL detectionsystem. For TCA precipitation, 100% (g/ml) TCA was added to each sampleto achieve a final concentration of 20%. The samples were kept on icefor 4 hours then the precipitate was collected by centrifugation.Pellets were washed twice with acetone and completely air dried beforeaddition of SDS sample buffer.

Electrophysiological Recordings and Statistical Analysis

Ventricular cardiomyocytes were cultured for two days. Whole-cellpatch-clamp recordings of L-VGCC currents were carried out fromspontaneously pulsing cardiomyocytes. The external solution was (in mM):145 TEACl, 9 BaCl₂, 0.5 MgCl₂, 5.5 glucose, 0.1 NiCl₂, and 5 HEPES, pH7.4 with CsOH or TEAOH. The pipette solution was (in mM): 125 Csacetate, 20 CsCl, 3MgCl₂, 10 EGTA, and 5 HEPES, pH 7.4 adjusted withCsOH. Currents were recorded at room temperature using an A-M Systemsmodel 2400 patch-clamp amplifier (Sequim, Wash., USA). Signals werelow-pass filtered at 2 kHz and digitized at 5 kHz with Digidata 1440Ainterface and pCLAMP 10.0 software (Molecular Devices, Sunnyvale,Calif., USA). Cardiomyocytes were held at −40 mV, and Ba²⁺ currents wererecorded immediately after whole-cell patches were formed by gentlesuctions. Current-voltage (I-V) relationships were elicited from aholding potential at −40 mV using 200 ms steps (5 seconds between steps)over a range from −60 to +60 mV in 10 mV increments. The currentdensities were calculated by dividing the current amplitudes (pA) bymembrane capacitances (pF).

Co-Immunoprecipitation and Proteomics with Mouse Brains

Adult mouse whole brains were collected and lysed in 8 mlimmunoprecipitation buffer containing a protease inhibitor cocktail. Thecell lysate was cleaned with 1 ml sepharose 4B resin prior to incubationwith peptide Lv antibody conjugated sepharose 4B. A 10 μg custom-maderabbit polyclonal antibody was immobilized in 40 μl AminoLink PlusCoupling Resin by following the manufacturer's protocol. The lysate andantibody were incubated for 5 hours at 4° C. The resins were washedthree times with immunoprecipitation buffer and twice with PBS. Sampleswere resolved on 12% SDS-PAGE gels and stained with Coomassie brilliantblue R-250. Four major bands ranging from 40 to 200 kD were carefullyexcised and subjected to in-gel digestion and matrix-assisted laserdesorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Massspectrometry and database analysis were performed in the Laboratory ofBiological Mass Spectrometry, Texas A&M University.

Co-Immunoprecipitation (Co-IP)

Eight chicken embryonic hearts (E18) were collected and homogenized in 4ml of immunoprecipitation buffer. Samples were rotated at 4° C. for 2hours to solubilize membrane proteins. Samples were then centrifuged toremove cell debris, and a small portion of the supernatant was taken forprotein quantification analyses and for a SDS-PAGE gel subsequentlystained with Coomassie brilliant blue R-250. The rest of the supernatantwas cleaned with Protein Agarose beads followed by incubating the beadswith 10 μl of the antibody (anti-peptide Lv or anti-VEGFR2) for 3 hours.Because both anti-peptide Lv and anti-VEGFR2 antibodies were derivedfrom the rabbit, rabbit IgG was used as the negative control. Anyprotein fractions that could co-IP with rabbit IgG, as well asanti-peptide Lv or anti-VEGFR2 antibody, would be considered to benon-specific protein targets of peptide Lv. After incubation, 20 μl ofProtein A-agarose was added to each tube and incubated for another 1.5hours. The beads were collected, washed, and processed for Westernblotting analysis of peptide Lv and VEGFR2. Western blots werevisualized as described previously. For some IPs, antibody preparatoryand immunoprecipitation kits were used to remove heavy and light chaininterference. All co-IPs were repeated three times.

Tetrazoliumdye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) Colorimetric Assay for Cell Proliferation

Human umbilical vein endothelial cells (HUVECs) and Human retinalendothelial cells (RECs) were seeded onto 24-well plates in endothelialcell growth medium and allowed to adhere overnight. The culture mediawere exchanged to opti-MEM for 45 minutes. VEGF or Peptide Lv at variousconcentrations were added to the cells, and the cells were continuouslyincubated for another 48 hours (HUVECs) or 96 hours (RECs). Opti-MEMwith 20% FBS alone acted as the negative control. The proliferation ofHUVECs was determined by the MTT assay following the manufacturer'sprotocol. In brief, cells were incubated with the MTT solution (1.2 mMfinal concentration) for 4 hours at 37° C. Then DMSO was added to afinal concentration of 50% in order to break the plasma membrane, andthe absorbance at 540 nm was measured by a spectrophotometer.

Assessment of Vasodilation Function

All animal procedures were performed in accordance with the Associationfor Research in Vision and Ophthalmology (ARVO) Statement for the Use ofAnimals in Ophthalmic and Vision Research and were approved by the Scottand White Institutional Animal Care and Use Committee. Pigs of eithersex (age range, 8-12 weeks; weight, 8-12 kg) purchased from Real Farms(San Antonio, Tex.) were sedated with Telazol (4.4 mg/kg,intramuscularly), anesthetized with 2-4% isoflurane, and intubated. Theeyes were enucleated and immediately placed into a moist chamber on ice.The anterior segment and vitreous body were removed carefully under adissecting microscope. The posterior segment or eyecup was placed in acooled dissection chamber (6° C.) containing a physiological saltsolution (PSS; in mM: NaCl 145.0, KCl 4.7, CaCl₂ 2.0, MgSO₄ 1.17,NaH₂PO₄ 1.2, glucose 5.0, pyruvate 2.0, EDTA 0.02, and MOPS 3.0) with 1%albumin. Retinal arterioles (<80 μm in diameter) were carefullydissected out and then transferred for cannulation to a Lucite vesselchamber containing PSS-albumin solution equilibrated with room air atambient temperature. One end of the arteriole was cannulated using aglass micropipette (tip outer diameter of 30-40 μm) filled withPSS-albumin solution, and the outside of the arteriole was securely tiedto the pipette with 11-0 ophthalmic suture. The other end of the vesselwas cannulated with a second micropipette and also secured with suture.After cannulation, the vessel and pipettes were transferred to the stageof an inverted microscope (Olympus CKX41) coupled to a video camera(Sony DXC-190, Labtek, Campbell, Calif.) and video micrometer(Cardiovascular Research Institute, Texas A&M University System HealthScience Center, College Station, Tex.) for continuous measurement of theinternal diameter throughout the experiment. The cannulating pipetteswere connected to independent pressure reservoirs. By adjusting theheight of the reservoirs, the vessel was pressurized to 55 cmH₂Ointraluminal pressure without flow. This level of pressure was usedbased on pressure ranges that have been documented in retinal arteriolesin vivo and in the isolated, perfused retinal microcirculation. Afterdeveloping resting vascular tone (˜40 min), the concentration-dependentresponse of isolated vessels to peptide Lv, (1 to 20 μg/ml) was examinedand the diameter changes were recorded. At the end of the experiments,the vessels were maximally dilated with sodium nitroprusside (0.1 mM) inthe bath containing zero calcium. These maximum dilations were used tonormalize the peptide Lv response and expressed as % maximum dilation.

In Ovo (In Vivo) Chicken Chorioallantoic Membrane (CCM) Assays forAngiogenesis and Vasculogenesis

The in ovo (in vivo) CCM assay is an effective test to determine whetherexogenous molecules promote blood vessel growth in length (angiogenesis)or forming new blood vasculature (vasculogenesis). Using shell-lesschicken embryo cultures, two coverslips coated with PBS (vehicle),peptide Lv (5 μg/per coverslip), or anti-peptide Lv (1 μg/per coverslip)were placed on the top of the CCM of each embryo at embryonic day 7 (E7)or E8. At E11 or E12, photographs of the CCM areas with coverslips weretaken, and the vasculature areas and vessel lengths were analyzed.

Intra-Ocular Injection and Retinal Vessel Staining

The male and female C57BL/6J mice were originally purchased from Harlan(Houston, Tex., USA). All animal experiments were approved by theInstitutional Animal Care and Use Committee of Texas A&M University andwere performed in compliance with the ARVO Statement for the Use ofAnimals in Ophthalmic and Vision Research. Mice were housed undertemperature and humidity-controlled conditions with 12:12 hourlight-dark cycles. All mice were fed with laboratory chow and water adlibitum. At postnatal day 7 (P7), young mice were anesthetized withisoflurane, and the intravitreal injections into the vitreous of the eyewith PBS (vehicle buffer) or peptide Lv were performed using a Hamiltonsyringe. One eye from each mouse was injected with peptide Lv (at 2μg/μl) at 0.5 or 1 μl, and the other eye was either injected with PBS orwithout injection. At P13, mice were sacrificed, and the eyes were fixedwith Zamboni's fixative for 2 hours at 4° C. The retinas were isolatedand stained overnight at 25° C. with FITC-conjugated Isolectin B4 in PBScontaining 0.1% TX-100 and 1 mM Ca²⁺. Following 2 hours of washes,retinas were cut on the peripheral edge and flat-mounted with thephotoreceptor side down onto microscope slides in ProLong Antifadereagents. Images were taken at 5x magnification on a Zeiss DigitalImaging Workstation, and whole retinal images were stitched togetherwith the Image Composite Editor.

Endothelial Migration (Wound Healing) Assay

The HUVECs were cultured on coverslips to 70% confluence. A “scratch”gap was generated using a 200 μl-pipette tip. Cultures were treated witheither PBS (control) or peptide Lv (200 or 800 ng/ml). Photos were takenwith the Olympus IX 71 inverted microscope at various time post-scratch,and cell migration distance from the scratch mark was measured (inpixels) using Adobe Photoshop software, and the migration rate wascalculated as pixels per day (24 hours).

Assessment of Pathological Angiogenesis Using the Mouse Oxygen-InducedRetinopathy (OIR) Model

The mouse OIR model has been widely used as a model for pathologicalangiogenesis. At postnatal day 7 (P7), young newborn pups were kept in ahyperoxia environment (75% oxygen) until P12, and after P12, the pupswere returned back to the normal room air (normoxia). Under hyperoxia(from P7 to P12), there is blood vessel loss in the retina. After P12 toP17 under normal room air (normoxia), retina will have robust re-growthof blood vessels, including angiogenesis, vasculogenesis, and vesselproliferation, so the OIR model is used to induce pathologicalangiogenesis. Testing molecules or vehicle (control) are injected intothe eyes at P12 immediately upon returning to the normal room air, andmice are sacrificed with the retinas excised at P17 for furtheranalyses. The retinal areas that are lacking vasculature (“avasculararea”) are quantified. If a molecule promotes angiogenesis, theavascular retinal area of the molecule-injected eye will be less thanthe control eye (injected with vehicle). If a molecule preventsangiogenesis, the avascular retinal area of the molecule-injected eyewill be larger than the control eye. At P12, one eye from each youngmouse was injected with PBS (vehicle), while the other eye was injectedwith anti-peptide Lv (2 μg/μl). At P17, mice were sacrificed, and theeyes were fixed with Zamboni's fixative for 2 hours at 4° C. The retinaswere isolated and processed for retinal vasculature staining asdescribed above, and the retinal area that was lacking vasculature(“avascular area”) were quantified.

RESULTS Peptide Lv Enhances L-VGCC Activities in Time- andDose-Dependent Manners in Cardiomyocytes

It has been shown that peptide Lv enhances the L-VGCC currents inretinal photoreceptors in time- and dose-dependent fashions. Since theL-VGCCs are essential in the excitation-contraction coupling ofcardiomyocytes, it was postulated that peptide Lv might also regulatethe L-VGCCs in cardiomyocytes similar to its action in thephotoreceptors. FIG. 1 shows that peptide Lv enhances L-VGCC currentsand protein expression in cultured embryonic cardiomyocytes; FIGS. 1Aand 1B show that the augmentation of L-VGCC currents by peptide Lv isconcentration-dependent. Cardiomyocytes were dissociated and cultured atE12, and L-VGCC currents were recorded at E14. Cultures were treatedwith 0, 500, and 1000 ng/ml of synthetic peptide Lv for 4 h prior toelectrophysiological recordings. There was a stepwise increase in theL-VGCC current densities with 1000 ng/ml peptide Lv being significantlyhigher. FIGS. 1C and 1D shows that the effect of peptide Lv on LVGCCs incardiomyocytes is time-dependent. Cardiomyocytes were treated withsynthetic peptide Lv (500 ng/ml) for 0, 30, or 60 min prior toelectrophysiological recordings. FIG. 1E shows that peptide Lv enhancesL-VGCCα1 expression. Treatment with synthetic peptide Lv (500 ng/ml) for4 h in cultured cardiomyocytes (E12+2) elicited a significant increaseof L-VGCCα1 expression (1.98±0.29 folds; * indicates a significantdifference at p<0.05). FIGS. 1F and 1G show that an anti-peptide Lvantibody (a-peptide Lv) decreases L-VGCC currents. Cultures were treatedwith peptide Lv antibody or heat-inactivated peptide Lv antibody (5μg/ml; denatured antibody) for 24 h prior to electrophysiologicalrecordings. Treatment with the peptide Lv specific antibody decreasedthe maximal current density of L-VGCCs compared to the L-VGCCs recordedfrom the control or cardiomyocytes treated with denatured antibody.

Cultured embryonic cardiomyocytes were treated with a synthetic peptideLv for 4 hours at 500 ng/ml or 1000 ng/ml followed by patch-clampelectrophysiological recordings of L-VGCC currents. At 1000 ng/ml,peptide Lv elicited significantly higher L-VGCC currents (e.g., seeFIGS. 1A and 1B). Treatment with peptide Lv for only 30 or 60 minutesquickly elicited an increase of L-VGCC currents (e.g., see FIGS. 1C and1D), which was in part through an increase of protein expression of thepore-forming L-VGCCα1 subunits (e.g., see FIG. 1E). Because the mRNA ofpeptide Lv is present in various tissues, including the heart, eye, andvarious brain areas, the existence of endogenous functional peptide Lvneeds to be verified. If a functional peptide Lv is expressed in theheart, application of an antibody specifically against peptide Lv wouldaffect L-VGCCs by antagonizing the action of endogenous peptide Lv inembryonic cardiomyocytes. Cultured embryonic cardiomyocytes were treatedwith a specific antibody against peptide Lv for 18-22 hours prior topatch-clamp recordings. Cardiomyocytes treated with the anti-peptide Lvantibody (a-peptide Lv) showed decreased L-VGCC currents, while incontrast, cardiomyocytes treated with a denatured anti-peptide Lvantibody did not have effect on L-VGCC currents (FIG. 1F). These resultsdemonstrated that exogenous peptide Lv augments the L-VGCCs by enhancingthe expression of L-VGCCα1 subunits, and blocking the endogenous peptideLv dampens the L-VGCC currents, indicating a functional role of peptideLv in cardiomyocytes.

Identification of VEGFR2 (KDR/FLK-1) as a Binding Partner for Peptide Lv

A proteomics approach was used to identify potential receptors orbinding partners in order to determine the underlying molecularmechanisms of peptide Lv on L-VGCCs in both photoreceptors andcardiomyocytes. Since the mouse brain also expresses peptide Lvabundantly and yields more tissue than the heart, a mouse whole brainpreparation with co-immunoprecipitation (co-IP) was used followed by aSDS-PAGE and mass spectrometry (MS) analysis to determine the potentialreceptor candidates for peptide Lv. Excluding the cytoskeleton chaperoneproteins (myosin, clathrin heavy chain, and tubulin), there were threepotential receptor-like candidates, including KDR protein(VEGFR2/KDR/FLK-1) (Access No. EDL37891), Fc receptor-like B (Access No.NP_001025155), and vomeronasal type-1 receptor (Access No. NP_035814).The vomeronasal type-1 receptor is a G-protein coupled pheromonereceptor mainly located in the olfactory bulb. The Fc receptor-like B isa member of the Fc receptor family that is involved in phagocytosis,antibody-dependent cell cytotoxicity, and transcytosis. The KDR/FLK1protein, also known as the vascular endothelial growth factor receptor 2(VEGFR2), belongs to the tyrosine kinase (TK) receptor family. Furtherproteomics analysis indicated that VEGFR2 (KDR/FLK1) and vomeronasaltype-1 receptor were the possible candidate receptors for peptide Lv. Todetermine which receptor interacted with peptide Lv, co-IP assays wereemployed, and an interaction between peptide Lv and VEGFR2 in the chickhearts was found. Using the anti-peptide Lv antibody, a protein near 250kD was coimmunoprecipitated from the E18 chicken hearts, which wasdetected with the anti-VEGFR2 antibody. While the anti-VEGFR2 antibodywas used to co-immunoprecipitate proteins, a protein near 6 kD wasdetected with the anti-peptide Lv antibody. In both cases, the presenceof VEGFR2 or peptide Lv was not detected using the rabbit IgG for co-IP.In cultured cardiomyocytes, treatment with a selective VEGFR2 inhibitor,DMH4, was able to block the augmentation effect of peptide Lv on L-VGCCcurrents (FIGS. 2A and 2B), but DMH4 by itself did not affect theLVGCCs, indicating that the action of peptide Lv on L-VGCCs was in partthrough the VEGFR2. Hence, VEGFR2 (KDR/FLK1) is a potential receptor forpeptide Lv.

Peptide Lv Stimulates VEGFR2 Autophosphorylation in Cardiomyocytes

Since VEGFR2 belongs to a tyrosine kinase receptor family that issubjected to autophosphorylation of tyrosine residues after the receptoris activated via ligand binding, whether peptide Lv could elicittyrosine phosphorylation of VEGFR2 was examined next. Among the fourtyrosine phosphorylation sites on VEGFR2, tyr1054/1059 phosphorylationis required for maximal kinase activation and is considered aprerequisite for activating VEGFR2 signaling. Because the amino acidsequences near tyr1054/1059 of VEGFR2 are highly conserved across thechicken, mouse, and human, the specific antibody against phosphorylatedVEGFR2 at tyr1054/1059 (pVEGFR2-tyr1054/1059) was used to detect VEGFR2autophosphorylation. A p-tyr-100 antibody that commonly used to detectphosphorylated tyrosine residues, was employed to assess the activationof tyrosine kinases. Cultured chicken embryonic cardiomyocytes weretreated with peptide Lv in the presence or absence of VEGFR2 inhibitorDMH4. Peptide Lv was found to enhance phosphorylation of VEGFR2(pVEGFR2-tyr1054/1059) and phosphorylated tyrosine in a manner sensitiveto DMH4 inhibition. Treatment with peptide Lv was able to elicittyrosine autophosphorylation of VEGFR2 (pVEGFR2-tyr1054/1059) within 30minutes, and this phosphorylation was time-dependent with a maximalactivation at 2 hours. Hence, VEGFR2 is a candidate receptor for peptideLv.

Peptide Lv Activates Downstream Signaling Molecules of VEGFR2 inCardiomyocytes

Activation of VEGFR2 triggers several downstream signaling cascades,including phosphoinositide phospholipase C (PLCγ), phosphoinositide3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) andextracellular signal-regulated kinase (ERK) pathways. Phosphoinositidephospholipase C (PLC) participates in phosphatidylinositol4,5-bisphosphate (PIP2) metabolism and generates inositol triphosphate(IP3) and diacylglycerol (DAG), in which DAG further serves as anactivator of protein kinase C (PKC). In both porcine aortic endothelialcells and cultured cardiomyocytes, activation of VEGFR2 causesphosphorylation and activation of ERK1/2. Since PKC and ERK aredownstream of VEGFR2, whether these signaling molecules would respond topeptide Lv was examined in a series of experiments. In culturedcardiomyocytes, treatment of peptide Lv for 4 hours increased thephosphorylation levels of ERK (pERK, FIG. 3A) and PKC (pPKCα/β). Whilethe general G-protein coupled receptor (GPCR) inhibitor SCH202676 didnot inhibit the effects of peptide Lv on pERK and pPKCα/β, the VEGFR2inhibitor DMH4 did, indicating that peptide Lv is able to elicitactivation/phosphorylation of the downstream signaling targets ofVEGFR2, such as the ERK and PKC. Treatment with PD98059 (50 μM), aninhibitor of MEK1 upstream of ERK, decreased the peptide Lv augmentationof L-VGCC currents (FIGS. 3B and 3C), as did the PKC inhibitorchelerythrine (2 μM, FIGS. 3D and 3E). Inhibition of PKC or MEK1-ERKsignaling blocked the effect of peptide Lv on L-VGCC activity. Hence,the action of peptide Lv on L-VGCC augmentation is suggested to bemediated by the VEGFR2 binding, which further activates downstreamsignaling, including PKC and ERK.

The VEGF Signaling Pathway Increases L-VGCC Activities in theCardiomyocytes

As discussed earlier, peptide Lv augments L-VGCC activities through theVEGFR2 signaling pathway in chicken cardiomyocytes. While both VEGFR2and VEGFa (an endogenous VEGFR2 agonist) are present in culturedembryonic or neonatal cardiomyocytes, thus far, there is no report onthe increase of L-VGCCs following the direct activation of VEGFR incardiomyocytes. Treatment with VEGFa (50 ng/ml) for 4 hourssignificantly increased L-VGCC currents (FIGS. 4A and 4B). Treatmentwith the VEGFR2 antagonist (DMH4,5 μM) blocked the augmentation ofL-VGCCs by VEGFa (FIGS. 4A and 4B). Treatment with a specific antibodyagainst VEGFa (VEGF antibody) significantly decreased the L-VGCCsactivity in cultured embryonic cardiomyocytes (FIGS. 4C and 4D),indicating that endogenous VEGF signaling is involved in the regulationof L-VGCCs in embryonic cardiomyocytes. These data support the notionthat activation of VEGFR2 can augment the L-VGCCs in cardiomyocytes,which further supports that peptide Lv mimics endogenous VEGF andincreases L-VGCC currents in cardiomyocytes through activation of VEGFR2signaling.

Peptide Lv Stimulates the VEGFR2 Activation in the Human Umbilical VeinEndothelial (HUVE) Cells

The interaction between peptide Lv and VEGFR2 was also tested incultured human umbilical vein endothelial cells (HUVECs) to verify thatthe action of peptide Lv to activate VEGFR2 was not solely in chickencardiomyocytes. HUVECs were chosen due to the high expression of VEGFR2in vascular endothelial cells and the identified role of VEGFR2signaling in angiogenesis. The VEGFR2 was co-immunoprecipitated (co-IP)with the anti-peptide Lv antibody (a-peptide Lv) but not with the rabbitIgG from cultured HUVECs, which was similar to the co-IP result inembryonic chick hearts. To further validate if peptide Lv could alsoactivate VEGFR2 (measured as pVEGFR2) in HUVECs as in chickencardiomyocytes, cultured HUVECs were treated with 500 ng/ml peptide Lvfor 0, 30, or 60 minutes before cells were harvested for Westernimmunoblotting. Antibodies against pVEGFR2 (tyr1054/1059), total VEGFR2,total ERK1/2, and pERK1/2 were used in the Western blots. Treatmentswith peptide Lv in cultured HUVECs increased both pVEGFR2 and pERK1/2levels. Therefore, peptide Lv is able to activate VEGFR2 and itsdownstream signaling pathway in cultured human endothelial cells, aswell as in chicken cardiomyocytes.

VEGF signaling regulates the proliferation, migration, and survival ofendothelial cells and thus promotes angiogenesis. Mutations of VEGFR2result in deficits in blood-island formation and angiogenesis, which islethal during embryonic development. In response to angiogenic stimuli,endothelial cells proliferate, migrate, and coalesce to form a primitivevascular system and further recruit smooth muscle cells to give rise tomature blood vessels. Since peptide Lv activates VEGFR2 and itsdownstream signaling in both cardiomyocytes and vascular endothelialcells, it is possible that peptide Lv might enhance the proliferation ofendothelial cells thus promoting angiogenesis. The HUVECs were treatedwith peptide Lv (200 or 500 ng/ml) or vehicle for 48 h, and thensubjected to the tetrazolium dye (MTT) colorimetric assay to assess cellproliferation. The cells treated with peptide Lv (both at 200 and 500ng/ml) had a significant increase in the color absorbance at 540 nm(FIG. 5), indicating that peptide Lv stimulates the proliferation ofendothelial cells.

Peptide Lv Elicits Vasodilation

The proper function of an organ requires adequate supply of blood flow(e.g., oxygen and nutrients) to the cells and tissues. The smallarterioles (<100 μm in diameter) play a critical role in the control ofblood flow by contracting or relaxing vascular smooth muscle, whichcauses vasoconstriction or vasodilation, respectively. Therefore, thedilation of arterioles becomes important in terms of mainlining tissueblood flow during ischemia or hypoxia. VEGF is known to be releasedduring tissue hypoxia or ischemia, and it can cause vasodilation toincrease blood flow. To determine whether peptide Lv also exhibitsvasomotor activity (vasodilation ability) like VEGF, porcine retinalarterioles were freshly isolated and then tested its reaction to peptideLv. Treatment with peptide Lv dilates the arteriole (FIG. 6A), and itsaction is similar to that of VEGF as a potent vasodilator. Thevasodilator property of peptide Lv can be utilized to reduce bloodpressure and promote perfusion of microcirculation and optimally enhancetissue oxygenation and survival under stress.

Peptide Lv and VEGF Work Synergistically in Promoting Endothelial CellProliferation

While peptide Lv is able to bind to VEGFR2 and elicit cellproliferation, it is not clear whether peptide Lv might compete withVEGF for VEGFR2, or have synergistic action with VEGF. The human retinalendothelial cells (RECs) were cultured and treated with VEGF with orwithout peptide Lv for up to 96 hr. The MTT assays were carried out tomeasure the proliferation of RECs. Treating RECs with low concentrationof VEGF (at 1 ng/ml) or peptide Lv (at 50 ng/ml) alone did not elicitsignificant proliferation in RECs compared to the control (treated withvehicle buffer). However, treating RECs with combined VEGF (1 ng/ml) andpeptide Lv (10 ng/ml or 50 ng/ml) significantly promoted the cellproliferation, compared to the control or VEGF (1 ng/ml) alone (FIG.6B), indicating that peptide Lv and VEGF work synergistically inpromoting endothelial proliferation. This is the first evidence showingthe synergistic interaction of peptide Lv and VEGF in endothelial cells.This unique interaction highlights the potential of using peptide Lv andits antagonists to manipulate angiogenesis and tissue repair underpathophysiological conditions.

Peptide Lv Elicits Angiogenesis and Neovascularization In Vivo andPromotes Wound Healing In Vitro

Even though peptide Lv promotes endothelial cell proliferation, which isthe essential step for angiogenesis and neovascularization, it was notknown whether peptide Lv is able to promote angiogenesis in animals.Direct injections of peptide Lv (2 μg/μl) into postnatal day 7 (P7)mouse eyes through intravitreal injections strikingly increased thegeneration of new blood vessels (neovascularization), compared to thecontrol (no injection) or injection with PBS (vehicle buffer). This isthe first direct evidence that peptide Lv is an angiogenic agentpromoting neovascularization in living tissues. In cultured HUVECs with70% confluence, a scratch gap was generated thus representing a scratch“wound injury”. Treatments with peptide Lv at 200 or 800 ng/mlsignificantly enhanced the endothelial cell migration rate compared tothe control (treated with vehicle buffer), suggesting that peptide Lv isable to promote wound healing (FIG. 6C).

In Ovo (In Vivo) Chicken Chorioallantoic Membrane (CCM) AssaysDemonstrate that Peptide Lv Promotes Angiogenesis and Vasculogenesis,while Anti-Peptide Lv Antibody (Anti-Peptide Lv) Dampens Angiogenesisand Causes Vaso-Obliteration

The in ovo (in vivo) CCM assay is an effective test to determine whetherexogenous molecules promote blood vessel growth in length (angiogenesis)or forming new blood vasculature (vasculogenesis). Using shell-lesschicken embryo cultures, two coverslips coated with either PBS (vehicle)or peptide Lv (5 μg/per coverslip) were placed on the top of the CCM ofeach embryo at embryonic day 7 (E7) or E8. At E11, more capillaries andsmall vascular branches are observed under the coverslips coated withpeptide Lv. Hence, peptide Lv is able to promote angiogenesis andvasculogenesis in chicken embryos in ovo (FIGS. 7A, 7B). Using a similarexperimental design with coverslips coated with either PBS oranti-peptide Lv (1 μg/per coverslip), it was found that the CCM areascovered with anti-peptide Lv for ˜4 days develop fewer capillaries andvascular branches compared to the areas covered with PBS-coatedcoverslips (control; FIGS. 7A, 7B)). Some small vessels underanti-peptide Lv showed signs of vaso-obliteration.

Intraocular Injections of Peptide Lv or Anti-Peptide Lv into EarlyPostnatal Young Mouse Eyes Demonstrate that Peptide Lv PromotesAngiogenesis and Vasculogenesis, but Anti-Peptide Lv DecreasesAngiogenesis and Causes Vaso-Obliteration in the Retinal VasculatureDuring Normal Development

The retinal vasculature is undergoing angiogenesis and vasculogenesisduring the first 3 weeks after birth. The postnatal day 7 (P7) mice weregiven a single intraocular injection into one eye with PBS (vehicle),and the other eye was injected with either peptide Lv (2 μg/μl), oranti-peptide Lv (2 μg/2 μl) for 7 days. At P14, the retinas wereprocessed and stained for retinal vasculature. The eyes injected withpeptide Lv have a significant increased retinal vasculature compared tothe eyes injected with PBS or without injection, while the eyes injectedwith anti-peptide Lv shows signs of vaso-obliteration and decreasedretinal vasculature.

Anti-Peptide Lv Prevents the Re-Growth of New Blood Vessels in the MouseOxygen-Induced Retinopathy (OIR) Model

The mouse OIR model has been widely used as a model for pathologicalangiogenesis. At postnatal day 7 (P7), young newborn pups are kept in ahyperoxia environment (75% oxygen) until P12. Under hyperoxia (from P7to P12), there is blood vessel loss in the retina. After P12 to P17under normal room air (normoxia), retina will have robust re-growth ofnew vessels, which indicates the pathological angiogenesis andneovascularization. Molecules are injected into the eyes at P12, and theretinas are analyzed at P17. If a molecule promotes angiogenesis, theretinal area that is lacking vasculature (avascular area) will be lessthan the control eye (injected with vehicle). If a molecule preventsangiogenesis, the avascular area will be larger than the control eye. AtP12, one eye from each young mouse was injected with PBS (vehicle),while the other eye was injected with anti-peptide Lv (2 μg/μl). Theeyes injected with anti-peptide Lv had a larger avascular area comparedto the eyes injected with PBS (FIG. 8). These data provide evidence thatanti-peptide Lv is able to dampen pathological angiogenesis in vivo.

Modified Peptide Lv, the Inverso D-Peptide Lv, has a Similar Effect onCell Proliferation Using the Tetrazoliumdye3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)Colorimetric Assay for Cell Proliferation

Human umbilical vein endothelial cells (HUVECs) were seeded onto 24-wellplates in endothelial cell growth medium and allowed to adhereovernight. The culture media were exchanged to opti-MEM for 45 minutes.Peptide Lv or inverso D-peptide Lv (the very first amino acid at theN-terminal inverted from the natural L-isomer to D-isomer) at variousconcentrations (10, 50, 100 ng/ml) were added to the cells, and thecells were continuously incubated for another 48 hours (HUVECs).Opti-MEM with 20% FBS alone acted as the negative control. The cellproliferation of HUVECs was determined by the MTT assay as describedpreviously. It was found that both peptide Lv and D-peptide Lv promoteHUVEC proliferation equally.

Anti-Peptide Lv Antibody has a Dose-Dependent Effect in BlockingVEGF-Elicited Endothelial Cell Proliferation in MTT Colorimetric Assaysfor Cell Proliferation

Using cultured HUVECs, cells were treated with various concentrations ofanti-peptide Lv antibody (Anti-P. Lv) in the presence (+) or absence (−)of VEGF (20 ng/ml). Anti-peptide Lv antibody clearly dampensVEGF-elicited cell proliferation in a dose-dependent manner (FIG. 9).

While the present invention has been described in terms of certainpreferred embodiments, it will be understood, of course, that theinvention is not limited thereto since modifications may be made tothose skilled in the art, particularly in light of the foregoingteachings.

What is claimed is:
 1. A method of promoting angiogenesis in a subject,the method comprising administering to the subject a compositioncomprising a portion of peptide Lv.
 2. The method of claim 1, whereinthe administration of the composition stimulates VEGF signaling.
 3. Themethod of claim 1, wherein the administration of the compositionpromotes vasodilation, angiogenesis, neovascularization, and L-VGCC'sactivities and function.
 4. The method of claim 1, wherein thecomposition promotes cardiovascular and neurological function, woundhealing, through promoting vasodilation, angiogenesis andlymphoangiogenesis, blood/lymphatic vessel repairing and new growth, andcardiac contractility and cardiac output, and enhancing cognitivefunction.
 5. A method of treating disease comprising administering to asubject in need thereof a composition comprising an anti-peptide Lvantibody.
 6. The method of claim 5, wherein the administration of theanti-peptide Lv antibody treats diseases that have pathologicalangiogenesis and lymphangiogenesis, L-VGCC hyperfunction, or neuraldysfunction.
 7. The method of claim 5, wherein the diseases comprisecancers, diabetic retinopathy, age-related macular degeneration, cardiacarrhythmia, and neuropathy.
 8. The method of claim 5, wherein theanti-peptide Lv antibody blocks an effect of peptide Lv, VEGF, or VEGFreceptors.
 9. A composition for promoting the growth of new bloodvessels, the composition comprising a modified peptide Lv, wherein thefirst amino acid at the N-terminal is inverted from an L-isomer to aD-isomer.
 10. The composition of claim 9, wherein the first amino acidis a modified aspartic acid.